CN113755423B - Application of embryonic cancerous cells in preparation of 3D cell culture matrix, preparation method and 3D cell culture matrix - Google Patents
Application of embryonic cancerous cells in preparation of 3D cell culture matrix, preparation method and 3D cell culture matrix Download PDFInfo
- Publication number
- CN113755423B CN113755423B CN202111187431.XA CN202111187431A CN113755423B CN 113755423 B CN113755423 B CN 113755423B CN 202111187431 A CN202111187431 A CN 202111187431A CN 113755423 B CN113755423 B CN 113755423B
- Authority
- CN
- China
- Prior art keywords
- cell culture
- cell
- cells
- hours
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000012604 3D cell culture Methods 0.000 title claims abstract description 71
- 238000002360 preparation method Methods 0.000 title claims abstract description 28
- 239000012598 cell culture matrix Substances 0.000 title claims abstract description 18
- 239000006143 cell culture medium Substances 0.000 claims abstract description 28
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims description 84
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 44
- 239000000758 substrate Substances 0.000 claims description 34
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 22
- 239000011780 sodium chloride Substances 0.000 claims description 22
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 18
- 239000004202 carbamide Substances 0.000 claims description 18
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 15
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 11
- 238000005119 centrifugation Methods 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 8
- 210000002889 endothelial cell Anatomy 0.000 claims description 6
- 206010006187 Breast cancer Diseases 0.000 claims description 5
- 208000026310 Breast neoplasm Diseases 0.000 claims description 5
- 206010043276 Teratoma Diseases 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 210000003606 umbilical vein Anatomy 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims 3
- 239000000243 solution Substances 0.000 claims 3
- 210000004204 blood vessel Anatomy 0.000 claims 1
- 210000004216 mammary stem cell Anatomy 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 13
- 201000011510 cancer Diseases 0.000 abstract description 10
- 241001465754 Metazoa Species 0.000 abstract description 6
- 244000052769 pathogen Species 0.000 abstract description 5
- 230000001717 pathogenic effect Effects 0.000 abstract description 5
- 230000017095 negative regulation of cell growth Effects 0.000 abstract description 2
- 238000000502 dialysis Methods 0.000 description 28
- 238000000034 method Methods 0.000 description 15
- 239000000047 product Substances 0.000 description 15
- 230000000408 embryogenic effect Effects 0.000 description 13
- 239000002609 medium Substances 0.000 description 11
- 230000004069 differentiation Effects 0.000 description 10
- 238000004113 cell culture Methods 0.000 description 8
- 239000002244 precipitate Substances 0.000 description 8
- 230000012010 growth Effects 0.000 description 7
- 238000004153 renaturation Methods 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 108010085895 Laminin Proteins 0.000 description 5
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 229920003023 plastic Polymers 0.000 description 5
- 206010039491 Sarcoma Diseases 0.000 description 4
- 230000002411 adverse Effects 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 210000002919 epithelial cell Anatomy 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 3
- 210000002469 basement membrane Anatomy 0.000 description 3
- 210000000481 breast Anatomy 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 108010008217 nidogen Proteins 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 102100037369 Nidogen-1 Human genes 0.000 description 2
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 2
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 229940126864 fibroblast growth factor Drugs 0.000 description 2
- 210000004293 human mammary gland Anatomy 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 210000000578 peripheral nerve Anatomy 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 229960000187 tissue plasminogen activator Drugs 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 238000012605 2D cell culture Methods 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 230000005541 medical transmission Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009772 tissue formation Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0062—General methods for three-dimensional culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0631—Mammary cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/069—Vascular Endothelial cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Reproductive Health (AREA)
- Gynecology & Obstetrics (AREA)
- Oncology (AREA)
- Vascular Medicine (AREA)
- Dermatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides application of embryonic cancerous cells in preparation of a 3D cell culture matrix, a preparation method and the 3D cell culture matrix, and relates to the technical field of biology. In addition, homologous embryonic cancer cells can be selected to prepare the 3D cell culture medium according to the subsequent culture requirement, so that the problems of inhibition of cell growth, complicated preparation and the like caused by animal heterologous protein and pathogen pollution are effectively avoided.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of embryonic carcinoma cells in preparation of a 3D cell culture medium, a preparation method and the 3D cell culture medium.
Background
Compared with 2D cell culture, the 3D cultured cells show a higher degree of intercellular interactions, have more physiologically relevant morphology, and can better reproduce higher order tissue processes. In recent years, stem cells and 3D cell culture systems are becoming key models for research of regenerative medicine, drug development, toxicology and cancer, have important significance in basic research and transformation medicine application, and have wide application prospects in the fields of regenerative medicine, disease models, personalized medicine screening, accurate medicine and the like.
Currently, the main product in the market, such as Matrigel basement membrane extract of BD company, is obtained by separating and purifying tumor sarcoma. Although BD substrate film can support cell layers and also affect cell adhesion, migration, proliferation and differentiation in tissue formation, such substrate film is a heterologous product, which is prone to other adverse effects such as cell growth inhibition due to animal heterologous proteins and pathogen contamination, and differences between product batches. In view of this, the present invention has been made.
Disclosure of Invention
It is a first object of the present invention to provide the use of embryogenic cells in the preparation of a 3D cell culture medium, to at least alleviate one of the technical problems of the prior art.
A second object of the present invention is to provide a method for preparing a 3D cell culture substrate.
A third object of the present invention is to provide a 3D cell culture substrate prepared by the above preparation method.
The invention provides application of embryonic cancerous cells in preparation of a 3D cell culture medium.
Further, the embryogenic cancerous cells are mouse teratoma cells.
The invention also provides a preparation method of the 3D cell culture matrix, which comprises the steps of precipitating a cell culture solution of embryonic cancerous cells by using ammonium sulfate, re-dissolving the obtained precipitate by urea, and taking a supernatant for dialysis and renaturation to obtain the 3D cell culture matrix.
Further, the embryogenic cancerous cells are single cells or cell lines, preferably mouse teratoma cell lines.
Further, the embryonic cancerous cells are cultured using a serum-free cell culture medium, preferably using a serum-free DMEM culture medium;
Preferably, the time of the cultivation is 18 to 24 hours.
Further, the ammonium sulfate precipitation comprises mixing in 10 to 30% wt/vol ammonium sulfate solution for 8 to 14 hours, preferably in 20% wt/vol ammonium sulfate solution for 12 hours;
Preferably, the temperature of the mixing is 4 ℃;
Preferably, ammonium sulfate precipitation is performed after removal of cell debris from the cell culture broth of the embryogenic cancerous cells.
Further, the urea reconstitution comprises dissolving the obtained precipitate in Tris-HCl buffer containing 2-4M urea and 20-150 mM NaCl for 8-14 hours, preferably dissolving the obtained precipitate in Tris-HCl buffer containing 2M urea and 50mM NaCl for 12 hours;
preferably, the Tris-HCl buffer has a concentration of 10-100 mM, a pH of 7.4, and preferably a concentration of 50mM;
Preferably, the obtained precipitate is concentrated and then subjected to urea reconstitution.
Further, the dialysis renaturation comprises primary dialysis, secondary dialysis and tertiary dialysis which are sequentially performed;
Preferably, dialysis is performed using a dialysis bag.
Further, the first dialysis comprises dialysis at 4 ℃ for 8-14 hours;
Alternatively, the secondary dialysis comprises dialysis in Tris-HCl buffer containing 20-150 mM NaCl for 8-14 hours, preferably containing 50mM NaCl;
preferably, the Tris-HCl buffer has a concentration of 10-100 mM, a pH of 7.4, and preferably a concentration of 50mM;
Alternatively, the three dialysis comprises dialysis in DMEM solution, the temperature of the dialysis being 4 ℃.
In addition, the invention also provides the 3D cell culture medium prepared by the preparation method.
Compared with the prior art, the invention has at least the following beneficial effects:
the inventor of the present invention found through experiments that embryogenic cancer cells secrete a high content of soluble basement membrane mixture during differentiation, the main components of which include: laminin, collagen IV, and the like, as well as transforming growth factor-b, fibroblast growth factor, tissue plasminogen activator, and the like. The substrate film mixture can automatically aggregate to generate bioactive substrate materials similar to mammalian cell substrate films at room temperature, so as to be used for preparing 3D cell culture substrates capable of meeting different requirements. In addition, homologous embryonic cancer cells can be selected to prepare the 3D cell culture medium according to the subsequent culture requirement, so that the problems of inhibition of cell growth, complicated preparation and the like caused by animal heterologous protein and pathogen pollution are effectively avoided.
According to the preparation method of the 3D cell culture medium, the target product can be obtained by performing ammonium sulfate precipitation, urea redissolution and dialysis renaturation on the cell culture solution of the embryonic cancerous cells, and the method is simple to operate, convenient to observe and low in cost.
Based on the beneficial effects, the 3D cell culture medium provided by the invention can effectively help the attachment and differentiation of various cells such as epithelial cells, can influence the protein expression level of the cells, supports the regeneration of peripheral nerves, the differentiation of the epithelial cells and the like, has the advantages of stable quality, difficult degradation and the like, and fills up the technological and product blank in the technical aspects of stem cells and 3D cell basal cultures at home and abroad.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of comparison of growth of human breast cells MCF-10A provided in Experimental example 1 in the 3D cell culture substrate (A) without the invention and the 3D cell culture substrate (B) with the invention;
FIG. 2 is a graph showing the results of a Human Umbilical Vein Endothelial Cell (HUVEC) vascularization experiment provided in Experimental example 2 of the present invention in comparison with the 3D cell culture substrate (A) without the present invention and the 3D cell culture substrate (B) with the present invention;
FIG. 3 is a graph showing the results of comparison of the growth of human breast cancer cells (MDA-MB-231) provided in Experimental example 3 in accordance with the present invention in the 3D cell culture substrate (A) without the present invention and in the 3D cell culture substrate (B) with the present invention in terms of spheres spheroid;
FIG. 4 is a graph showing the results of the polyacrylamide gel electrophoresis analysis of the 3D cell culture medium of the invention and other similar products as provided in Experimental example 4.
Detailed Description
Unless defined otherwise herein, scientific and technical terms used in connection with the present application shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extraneous definition. In the present application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms is not limiting.
Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The present inventors have found that the preparation of a 3D cell culture medium using embryogenic cancerous cells has superior properties by extensive and intensive studies with respect to the problems associated with the preparation of a 3D cell culture medium using tumor sarcoma cells in the prior art, and completed the present invention on this basis.
According to a first aspect of the present invention there is provided the use of embryogenic cancer cells in the preparation of a 3D cell culture medium.
The present inventors have found that an embryogenic cancer cell, which is a malignant tumor cell formed in early stages of embryonic development, has various differentiation potential similar to stem cells, can secrete a high content of a soluble basement membrane mixture during differentiation, and has a major component including laminin, collagen IV, etc., which facilitates cell attachment and differentiation, and also contains transforming growth factor-b, fibroblast growth factor, tissue plasminogen activator, etc., by utilizing this characteristic of embryogenic cancer cells. The substrate film mixture can automatically aggregate to generate bioactive substrate materials similar to mammalian cell substrate films at room temperature, so as to be used for preparing 3D cell culture substrates capable of meeting different requirements.
In addition, in the practical use process, homologous embryonic cancerous cells can be selected according to the subsequent culture requirement to prepare the 3D cell culture matrix, for example, when the cells to be 3D cultured are murine cells, the embryonic cancerous cells which are murine cells can be correspondingly selected when the embryonic cancerous cells are selected, for example, but not limited to, mouse teratoma cells. Based on the method, other adverse effects such as cell growth inhibition, large batch-to-batch difference, complicated manufacture, high production cost, long preparation period and the like caused by animal heterologous protein and pathogen pollution can be effectively avoided.
According to a second aspect of the present invention, there is provided a method for preparing a 3D cell culture medium, comprising precipitating a cell culture solution of embryogenic cancerous cells using ammonium sulfate, redissolving the obtained precipitate with urea, and subjecting the supernatant to dialysis renaturation to obtain the 3D cell culture medium.
The preparation method is simple and feasible to operate, replaces the traditional tumor sarcoma separation and purification technical method, is simple to operate and convenient to observe, can effectively avoid cell growth inhibition caused by animal heterologous protein and pathogen pollution based on the traditional tumor sarcoma separation and purification technical method, has large batch-to-batch difference, complicated manufacture, high production cost, long preparation period and other adverse effects, has low requirements on the professional properties of technicians, and is suitable for popularization and application.
In some preferred embodiments, the embryogenic cancerous cell is a single cell or cell line.
The single cells or cell lines formed by single cell proliferation are selected as the embryonic cancer cells, so that the batch difference between 3D cell culture matrixes prepared by applying the embryonic cancer cells can be minimized, and the problem of the product batch difference in the prior art can be effectively solved.
In some preferred embodiments, the embryonic cancerous cells are cultured using a serum-free cell culture broth, preferably a serum-free DMEM culture broth.
Compared with the traditional culture medium, the culture method not only can meet the requirement of long-time in-vitro culture of the cells, but also can avoid adverse factors brought by animal serum, such as poor repeatability, high risk of disease transmission and immune response, and the like.
The time for culturing the embryogenic cancerous cells using the serum-free cell culture solution is 18 to 24 hours, and may be, for example, but not limited to, 18 hours, 20 hours, 22 hours, or 24 hours.
In some preferred embodiments, the ammonium sulfate precipitation comprises mixing in a 10-30% wt/vol ammonium sulfate solution for 8-14 hours, such as, but not limited to, 10% wt/vol, 15% wt/vol, 20% wt/vol, 25% wt/vol, or 30% wt/vol, for a period of time such as, but not limited to, 8 hours, 10 hours, 12 hours, or 14 hours. Wherein, the mixing can be performed by stirring.
Preferably, the temperature of the mixing is 4 ℃, and the precipitation operation is carried out under the condition of 4 ℃, so that the precipitation efficiency can be ensured on the basis of ensuring the activity of the effective components.
Preferably, ammonium sulfate precipitation is performed after removal of cell debris from the cell culture broth of the embryogenic cancerous cells. Through removing impurities such as cell fragments, the purity of the product can be further improved, and the subsequent 3D cell culture is facilitated.
In some preferred embodiments, the urea reconstitution comprises dissolving the resulting precipitate in Tris-HCl buffer containing 2-4M urea and 20-150 mM NaCl for 8-14 hours, wherein the urea content may be, for example, but not limited to, 2M, 3M or 4M, the NaCl content may be, for example, but not limited to, 20mM, 40mM, 60mM, 80mM, 100mM, 120mM or 150mM, and the mixing time may be, for example, but not limited to, 8 hours, 10 hours, 12 hours or 14 hours, preferably 12 hours.
The specific redissolution is selected to redissolve the precipitate, so that the solubility of the substrate is high, and the renaturation effect is good.
Preferably, the Tris-HCl buffer has a concentration of 10-100 mM, for example, but not limited to, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM, preferably 50mM, and pH 7.4;
preferably, the obtained precipitate is concentrated and then subjected to urea redissolution, and the redissolution after concentration can effectively improve the redissolution efficiency and reduce the impurity interference.
In some preferred embodiments, the dialysis renaturation comprises a primary dialysis, a secondary dialysis, and a tertiary dialysis performed sequentially.
The three times of dialysis can better improve the renaturation efficiency of the substrate protein and prevent the protein from being denatured.
Wherein, the primary dialysis comprises dialysis at 4 ℃ for 8-14 hours, such as, but not limited to, 8 hours, 10 hours, 12 hours or 14 hours;
the secondary dialysis comprises dialysis in Tris-HCl buffer containing 20-150 mM NaCl for 8-14 hours, the NaCl content may be, for example, but not limited to, 20mM, 40mM, 60mM, 80mM, 100mM, 120mM or 150mM, preferably 50mM NaCl, for a period of time such as, but not limited to, 8 hours, 10 hours, 12 hours or 14 hours;
Preferably, the Tris-HCl buffer has a concentration of 10-100 mM, for example, but not limited to, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM, preferably 50mM, and pH 7.4;
The three dialysis included dialysis in DMEM solution, the temperature of the dialysis being 4 ℃.
According to a third aspect of the present invention, there is provided a 3D cell culture substrate prepared using the above preparation method.
Based on the beneficial effects of the application and the preparation method, the 3D cell culture matrix provided by the invention can effectively help the attachment and differentiation of various cells such as epithelial cells, can influence the protein expression level of the cells, supports the regeneration of peripheral nerves, the differentiation of the epithelial cells and the like, has the advantages of stable quality, difficult degradation and the like, and fills the technological and product blank in the technical aspects of stem cells and 3D cell substrate cultures at home and abroad.
The invention is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.
Example 1
The embodiment provides a preparation method of a 3D cell culture substrate, which comprises the following steps:
The F9 cell line was placed in approximately 40 plastic tissue culture plates of 150cm 2 and incubated in serum-free DMEM medium for 18-24 hours at 37℃with 5% CO 2, after which the medium was collected by centrifugation, clarified cell debris and stirred overnight at 4℃in 20% (wt/vol) ammonium sulfate solution. The precipitated protein was concentrated by centrifugation, dissolved in 2M urea and 50mM NaCl,50mM Tris-HCl buffer (pH 7.4) and stirred for 12 hours, then the supernatant was centrifuged and dialyzed overnight at 4℃and then dialyzed against 50mM Tris-HCl,50mM NaCl buffer (pH 7.4) for 12 hours and finally against DMEM solution at 4 ℃. After the completion, the product was sub-packaged and frozen at-80 ℃.
Example 2
The embodiment provides a preparation method of a 3D cell culture substrate, which comprises the following steps:
The F9 cell line was placed in approximately 40 plastic tissue culture plates of 150cm 2 and incubated in serum-free DMEM medium for 18-24 hours at 37℃with 5% CO 2, after which the medium was collected by centrifugation, clarified cell debris and stirred overnight at 4℃in 10% (wt/vol) ammonium sulfate solution. The precipitated protein was concentrated by centrifugation, dissolved in 2M urea and 150mM NaCl,10mM Tris-HCl buffer (pH 7.4) and stirred for 12 hours, then the supernatant was centrifuged and dialyzed overnight at 4℃and then dialyzed against 100mM Tris-HCl,20mM NaCl buffer (pH 7.4) for 12 hours and finally against DMEM solution at 4 ℃. After the completion, the product was sub-packaged and frozen at-80 ℃.
Example 3
The embodiment provides a preparation method of a 3D cell culture substrate, which comprises the following steps:
The F9 cell line was placed in approximately 40 plastic tissue culture plates of 150cm 2 and incubated in serum-free DMEM medium for 18-24 hours at 37℃with 5% CO 2, after which the medium was collected by centrifugation, clarified cell debris and stirred overnight at 4℃in 30% (wt/vol) ammonium sulfate solution. The precipitated protein was concentrated by centrifugation, dissolved in 4M urea and 20mM NaCl,100mM Tris-HCl buffer (pH 7.4) and stirred for 12 hours, then the supernatant was centrifuged and dialyzed overnight at 4℃and then dialyzed against 10mM Tris-HCl,150mM NaCl buffer (pH 7.4) for 12 hours and finally against DMEM solution at 4 ℃. After the completion, the product was sub-packaged and frozen at-80 ℃.
Comparative example 1
The comparative example provides a method for preparing a 3D cell culture substrate, comprising:
The F9 cell line was placed in approximately 40 plastic tissue culture plates of 150cm 2 and incubated in serum-free DMEM medium for 18-24 hours at 37℃with 5% CO 2, after which the medium was collected by centrifugation, clarified cell debris and stirred overnight at 4℃in 8% (wt/vol) ammonium sulfate solution. The precipitated protein was concentrated by centrifugation, dissolved in 5M urea and 15mM NaCl,120mM Tris-HCl buffer (pH 7.4) and stirred for 12 hours, then the supernatant was centrifuged and dialyzed overnight at 4℃and then dialyzed against 120mM Tris-HCl,15mM NaCl buffer (pH 7.4) for 12 hours and finally against DMEM solution at 4 ℃. After the completion, the product was sub-packaged and frozen at-80 ℃.
Comparative example 2
The comparative example provides a method for preparing a 3D cell culture substrate, comprising:
The F9 cell line was placed in approximately 40 plastic tissue culture plates of 150cm 2 and incubated in serum-free DMEM medium for 18-24 hours at 37℃with 5% CO 2, after which the medium was collected by centrifugation, clarified cell debris and stirred overnight at 4℃in 20% (wt/vol) ammonium sulfate solution. The precipitated protein was concentrated by centrifugation, dissolved in 2M urea and 50mM NaCl,50mM Tris-HCl buffer (pH 7.4) and stirred for 12 hours, then the supernatant was centrifuged and transferred to 50mM Tris-HCl,50mM NaCl buffer (pH 7.4) and dialyzed for 12 hours. After the completion, the product was sub-packaged and frozen at-80 ℃.
In order to facilitate the differentiation of the schemes provided in the examples of the present invention and comparative examples, the differences of the examples are listed in the following table:
Experimental example 1
Comparison of 10 days of growth of human mammary gland cell MCF-10A acinus in 3D cell culture substrate (a) without this invention and 3D cell culture substrate (B) provided in example 1 of the invention.
The 3D cell culture substrate suspension provided in example 1 of the present invention was previously fixed in an 8-chamber slide culture dish, MCF-10A cells were then cultured in the 8-chamber slide culture dish, the MCF-10A cell culture was allowed to grow for 10 days, and then the cells were washed and fixed and stained with ethidium bromide, and examined for acinar morphogenesis under Olympic fluorescence microscopy (rhodamine fluorescence filter). While using the 3D cell culture medium without invention for comparison.
The results show that the human breast cell MCF-10A acinus cultured using the 3D cell culture medium provided in example 1 of the present invention can grow into a more complete morphology compared to the human breast cell MCF-10A acinus cultured without the 3D cell culture medium provided in example 1 of the present invention.
Experimental example 2
Comparison of Human Umbilical Vein Endothelial Cells (HUVEC) vascularization tubular network experiments in 3D cell culture matrices (a) without this invention and 3D cell culture matrices (B) provided in example 1 of the invention.
50 Μl of the 3D cell culture medium provided in example 1 of the present invention was placed in the corresponding well of a 96-well plate, gelled after incubation at 37℃for 30 minutes, human Umbilical Vein Endothelial Cells (HUVECs) were inoculated on the 96-well plate and cultured in Endothelial Cell Growth Medium (ECGM) (cell applied), initial density of 5X 10 4 cells/well, incubation at 37℃for 4-6 hours with 5% CO 2, and then capillary-like structures were observed with an Olympic inverted microscope. The results were compared to the pre-plating of the 3D cell culture matrix in 96 wells without the invention.
The results show that the human umbilical vein endothelial cells cultured by using the 3D cell culture medium provided in example 1 of the present invention promote angiogenesis more clearly and completely in the formed tubular network than the 3D cell culture medium provided in example 1 of the present invention.
Experimental example 3
Human breast cancer cells (MDA-MB-231) were compared for spheroid spheroid growth in a 3D cell culture substrate (A) without the invention and in a 3D cell culture substrate (B) provided in example 1 of the invention.
MDA-MB-231 breast cancer cells were inoculated in 96 wells at 3000 cells/well culture with a 3D cell culture medium suspension mix (10%) provided in example 1 of the present invention, incubated at 37℃for 72 hours with 5% carbon dioxide, induced, and static culture spheres formed. The medium was replaced with fresh medium at 24 hours intervals. The spheres were photographed with an Olympic inverted microscope after formation and image analysis was performed using imageJ software.
The results indicate that human breast cancer cells cultured using the 3D cell culture medium provided in example 1 of the present invention proliferated to form a complete sphere spheroid structure, compared to the 3D cell culture medium not provided in example 1 of the present invention.
Experimental example 4
Polyacrylamide gel electrophoresis and analysis with coomassie brilliant blue staining the 3D cell culture matrices provided in example 1 of the present invention (columns 1-3) and other like products (columns 4-6): it can be seen that the 3D cell culture substrate 170KD provided in example 1 of the present invention has laminin Entactin (shown in FIG. 4).
The results show that the 3D cell culture medium provided by the embodiment 1 of the invention contains more nidogen proteins (Entactin), can be connected with laminin and IV type collagen, and forms a three-level compound with the core of laminin and proteoglycan, thereby being more beneficial to the growth, attachment and differentiation of various cells.
Experimental example 5
Human mammary gland cell MCF-10A acinar culture experiments performed on the 3D cell culture matrices provided in examples 1-3 and comparative examples 1-2 of the present invention, the area value of each acinar structure was measured using an olybach microscope and ImageJ software, and the average acinar particle size value was calculated by pasting it into an excel spreadsheet, and the results are shown in the following table:
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (1)
1. Application of embryonic cancerous cells in preparing a 3D cell culture medium;
the embryonic cancerous cells are mouse teratoma cells F9;
The preparation method of the 3D cell culture matrix comprises the following steps: culturing a mouse teratoma cell F9 cell strain in serum-free DMEM culture solution for 18-24 hours at 37 ℃ under the condition of 5% CO 2, centrifuging and collecting culture medium, clarifying cell fragments, and stirring in 10-30% wt/vol ammonium sulfate solution at 4 ℃ for overnight; concentrating the precipitated protein by centrifugation, dissolving in 2-4M urea and 20~150mM NaCl,10~100 mM Tris-HCl buffer solution, stirring for 8-14 hours, centrifuging to obtain supernatant, dialyzing overnight at 4 ℃ in 2-4M urea and 20~150mM NaCl,10~100 mM Tris-HCl buffer solution, dialyzing for 8-14 hours in 20-150 mM Tris-HCl,10~100mM NaCl buffer solution, and dialyzing at 4 ℃ in DMEM solution to obtain the 3D cell culture substrate;
The 3D cell culture matrix is used for 3D culturing human mammary cells to form acini, and/or,
The 3D cell culture medium is used for 3D culturing human umbilical vein endothelial cells to form blood vessels, and/or,
The 3D cell culture matrix is used for 3D culturing human breast cancer cells to form a spherical structure.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111187431.XA CN113755423B (en) | 2021-10-12 | 2021-10-12 | Application of embryonic cancerous cells in preparation of 3D cell culture matrix, preparation method and 3D cell culture matrix |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111187431.XA CN113755423B (en) | 2021-10-12 | 2021-10-12 | Application of embryonic cancerous cells in preparation of 3D cell culture matrix, preparation method and 3D cell culture matrix |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113755423A CN113755423A (en) | 2021-12-07 |
| CN113755423B true CN113755423B (en) | 2024-04-26 |
Family
ID=78799283
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111187431.XA Active CN113755423B (en) | 2021-10-12 | 2021-10-12 | Application of embryonic cancerous cells in preparation of 3D cell culture matrix, preparation method and 3D cell culture matrix |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113755423B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5639616A (en) * | 1993-11-10 | 1997-06-17 | Arch Development Corporation | Isolated nucleic acid encoding a ubiquitous nuclear receptor |
| JP2004135519A (en) * | 2002-10-15 | 2004-05-13 | Japan Science & Technology Agency | Parietal-endoderm-like cell line and method for establishing the same and method for producing secretory protein |
| CA2457296A1 (en) * | 2003-08-19 | 2005-02-19 | Takahiro Ochiya | Methods for inducing differentiation of pluripotent cells |
| JP2006304668A (en) * | 2005-04-27 | 2006-11-09 | Japan Science & Technology Agency | F9 embryonic tumor cell usable in production of protein and utilization thereof |
| CN102586176A (en) * | 2012-01-11 | 2012-07-18 | 中国科学院生物物理研究所 | Novel animal source-free and feed layer-free human pluripotent stem cell culture system |
| JP2016190802A (en) * | 2015-03-31 | 2016-11-10 | Jsr株式会社 | Novel polymer, coating agent, cell culture substrate, cell culture method, and method for synthesis of polymer |
| CN107475188A (en) * | 2017-09-25 | 2017-12-15 | 广东颜值科技有限公司 | A kind of cultural method of cell culture medium and embryonic stem cell |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1730265B1 (en) * | 2004-03-24 | 2016-02-17 | Marisa E. E. Jaconi | 3d-cardiac tissue engineering for the cell therapy of heart failure |
| US8574904B2 (en) * | 2007-01-22 | 2013-11-05 | Organ Technologies Inc. | Method for production of mesenchymal cell, method for production of tooth, and mesenchymal cell for formation of tooth |
-
2021
- 2021-10-12 CN CN202111187431.XA patent/CN113755423B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5639616A (en) * | 1993-11-10 | 1997-06-17 | Arch Development Corporation | Isolated nucleic acid encoding a ubiquitous nuclear receptor |
| JP2004135519A (en) * | 2002-10-15 | 2004-05-13 | Japan Science & Technology Agency | Parietal-endoderm-like cell line and method for establishing the same and method for producing secretory protein |
| CA2457296A1 (en) * | 2003-08-19 | 2005-02-19 | Takahiro Ochiya | Methods for inducing differentiation of pluripotent cells |
| JP2006304668A (en) * | 2005-04-27 | 2006-11-09 | Japan Science & Technology Agency | F9 embryonic tumor cell usable in production of protein and utilization thereof |
| CN102586176A (en) * | 2012-01-11 | 2012-07-18 | 中国科学院生物物理研究所 | Novel animal source-free and feed layer-free human pluripotent stem cell culture system |
| JP2016190802A (en) * | 2015-03-31 | 2016-11-10 | Jsr株式会社 | Novel polymer, coating agent, cell culture substrate, cell culture method, and method for synthesis of polymer |
| CN107475188A (en) * | 2017-09-25 | 2017-12-15 | 广东颜值科技有限公司 | A kind of cultural method of cell culture medium and embryonic stem cell |
Non-Patent Citations (2)
| Title |
|---|
| 三维共培养体系促进去分化的软骨细胞再分化的实验研究;张廷帅;邹健宇;陈汉政;刘日许;郑仕聪;陈艺;张姝江;姚咏嫦;;中华关节外科杂志(电子版)(06);第693-698页 * |
| 腺病毒介导的Hes1基因抑制肝细胞癌细胞的增殖与迁移;于兵;尹刚;胡以平;;肿瘤防治研究;47(03);第22-27页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113755423A (en) | 2021-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | High cell density mixotrophic culture of Spirulina platensis on glucose for phycocyanin production using a fed-batch system | |
| CN114317443B (en) | Breast cancer organoid culture solution, and culture reagent combination and culture method thereof | |
| CN114292816B (en) | Lung cancer organoid culture solution, and culture reagent combination and culture method thereof | |
| CN111925982A (en) | Human bone marrow mesenchymal stem cell culture process applying three-dimensional cells | |
| CN110804587A (en) | Method for modeling production of mesenchymal stem cells by adopting improved microcarrier cell culture method | |
| CN105969720A (en) | Human vascular endothelial cell culture solution and culture method | |
| CN110564682A (en) | Method for large-scale production of human adipose-derived mesenchymal stem cell exosomes | |
| CN115322964B (en) | Construction method of 3D culture amniotic mesenchymal stem cell seed bank | |
| CN101831403B (en) | Method for amplifying mesenchymal stem cells of human umbilical cord and placenta in vitro | |
| Green et al. | Production of hyaluronate and collagen by fibroblast clones in culture | |
| US5489261A (en) | Hydrogels capable of supporting cell growth | |
| CN113755423B (en) | Application of embryonic cancerous cells in preparation of 3D cell culture matrix, preparation method and 3D cell culture matrix | |
| CN113736737B (en) | Primary glioma-related fibroblast culture method | |
| CN115261302B (en) | Matrigel and preparation method and application thereof | |
| WO2022213704A1 (en) | High-migration mesenchymal stem cell, and preparation method therefor and application thereof | |
| CN108034634B (en) | Method for separating endometrial mesenchymal stem cells from menstrual blood | |
| WO2021104360A1 (en) | Method for improving efficiency of in vitro suspension culture of human multifunctional stem cells | |
| CN115141760A (en) | Inonotus obliquus fermentation extract, preparation method thereof and application thereof in preparation of cosmetics | |
| CN105779375A (en) | Novel serum-free culture medium | |
| CN111607553B (en) | Ganoderma lucidum mycelium culture medium for high yield of polysaccharide and culture method thereof | |
| CN111793568B (en) | Pichia pastoris and application thereof | |
| Mendonça et al. | High density Vero cell culture on microcarriers in a cell bioreactor | |
| Melani et al. | A possible role of phosphate in regulating phosphatase-level in the rat kidney | |
| CN116836933B (en) | Liver and gall cancer organoid culture solution, culture reagent combination and culture method | |
| CN113502258B (en) | Method for obtaining pancreatic precursor cells and pancreatic beta cells by differentiation of human pluripotent stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |